Chronic mild hyperhomocysteinemia has been implicated as a risk factor for occlusive vascular disease and genetic variation in folate metabolism has been implicated in the development of this condition. The specific aims of this proposal are: 1) To screen for polymorphisms in genes encoding folate-dependent enzymes involved in homocysteine remethylation and to ascertain which of these polymorphisms potentially contribute to hyperhomocysteinemia in the general population. Genetic differences in DNA samples from subjects at the upper range of plasma homocysteine and those at the lower end of the normal range will be ascertained for the human methionine synthase (MS), methylenetetra-hydrofolate reductase (MTHFR), cytosolic and mitochondrial serine hydroxymethyltransferase (cSHMT, mSHMT) and folypolyglutamate synthetase (FPGS) genes. Simple genetic screening tests will be developed for any polymorphisms associated with hyperhomocysteinemia. 2) To determine the metabolic effects of genetic variations in these genes. The modified proteins will be expressed and purified and their properties characterized. The effects of their expression in tissue culture cells on the regulation of the homocysteine remethylation cycle and on other metabolic cycles of one carbon metabolism will be evaluated. 3) To study the pathological and metabolic effects of disruption of the mouse methionine synthase gene. Mice heterozygous (and possibly homozygous) for a MS knockout will be generated and the effects of the gene disruption on homocysteine levels and remethylation rates, and on fluxes through other cycles of one carbon metabolism, will be ascertained. These animals will also be a valuable and unique model for pernicious anemia allowing an investigation of disturbances of nucleotide and DNA synthesis associated with the condition. The long term goals of the project are to delineate the role of genetic heterogeneity in the development of hyperhomocysteinemia and to understand the mechanisms by which homocysteine hemostasis is regulated. (End of Abstract)